Nowadays, there are many biological control techniques have been invented for controlling insects and plants (Smith and Borstel, 1972. Science. 178. 1164-1174). A method invented to control of insect populations is named the “sterile insect technique” SIT. This method is also known with a different name as “sterile insect release method” SIRM. A model of the SIRM based on population parameters obtained from a large scale experiment to eradicate melon fly was first developed by Ito (1977. Appl. Ent. Zool. 12:310-312; 1979. Res. Popul. Ecol. 20. 216-226). The SIT method creates sterile male insects and releases them into natural habitats where the males would look for natural females to mate. These females would be sterile or to produce offspring that cannot develop up to the harmful stages. When a huge number of sterile males released in a chronic time, it can cause a collapse of the natural insect populations or even extinction. However, the insect created by the SIT method need to be undergone a sexing step to remove females. The reason is that in many insect species, even sterile females, if being released, they would look for blood meals and may transmit diseases (in mosquitoes) or damage fruits (in med fly). As such, release of the female insects must be avoided.
Currently, there are different methods to separate male insects from a total based on the differences of sexual traits as body sizes or eclosion time. However, systematic errors of those are high and more or less depending on each species, for instance, our data indicated that 8-15% females of Aedes aegypti mosquitoes can be misidentified as males (K. P. Hoang, unpublished data). Alternatively, insects can be frozen down on ice to separate females and males. However, this method is too high labor costs and can damage small insects as male mosquitoes.
In another approach, X and gamma rays are used to translocate a chromosome fragment which carries genes encoding for different colors of silk worm male and female eggs (Strunnikov, 1979. Theor. Appl. Genet. 55, 17-21; Strunnikov. 1983. Control of silkworm Reproduction, Development and Sex. MIR Publishers. Moscow). However, the mutant strains created by radiation are usually accompanied with a significant decline in male mating competitiveness in comparison with its wild type males. This can result in failures in vector control strategies if applying for the release of insect males. Besides, irradiation method is not specific to the certain target, the radiation not only causes big mutations in chromosome systems of the target organisms but can also be dangerous for producers. This is also an expensive method with plenty of limitations.
Asburner et al., (1998) disclose a method by introduction of an exogenous DNA fragment into the insect genetic system to create insect transgenic species (Insect Molecular Biology, 7 (3), 201-213). This approach was lately improved by a patent of Handler (2006. PN: U.S. Pat. No. 7,005,296B1).
DeVault disclose to use a female specific promoter to be ligated into a lethal gene. The gene is only activated in females and therefore males are uniquely remained in the selection. These males are irradiated for sterilization before releasing into nature (DeVault et al., 1996; Biotechnology, Vol 14; 46-49; DeVault et al., 1996. Genome Res. 6: 571-579). This achievement gains a big progress in the genetic sexing experiments, however the use of radiation can severely damage for small insects with its consequences of decreasing male mating competitiveness ability.
To avoid the radiation damages, a new method named RIDL (Release of Insects carrying a dominant lethal) has been disclosed in a patent (Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007. Area-Wide Control of Insect Pests: From Research to Field Implementation, Springer, Dordrecht, The Netherlands). The RIDL offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes as mentioned above. RIDL differs from conventional SIT in that the released insects are not sterilized by irradiation but its sterility is resulted from a homozygote for a dominant lethal gene. Highly efficient repressible RIDL systems were first demonstrated in Drosophila models and recently in the Mediterranean fruit fly (Thomas et al., 2000. Science, 287: 2474-2476; Gong et al., 2000. Nature Biotech., 23: 453-456.). This system exploits a tetracycline-repressible transactivator (tTA) to control expression of the dominant lethal (Gossen and Bujard, 1992. Proc Natl Acad Sci USA 1992, 89(12):5547-5551). The tetracycline (Tet) that to be mixed in larval rearing medium or food can bind to tTA and preventing it binding to tetO sequences and driving the effector gene. The tetO sequences plays a role of an operator would be free to suppress the lethal gene. In the absence of Tet, tTA protein binds to the operator sequence and the effector gene would be free to express. In natural environment where Tet is absent, released transgenic males mates with wild type females and their offspring would be killed by the effector gene activation. In Aedes mosquitoes, RIDL has been proved to be efficient, of which the males created haven't been declined their fitness when competing with wild type males. (Phuc et al., 2007; BMC Biology http://www.biomedcentral.com/1741-7007/5/11). However, the RIDL method has a serious shortcoming that it still produces offspring in both sexes. It therefore needs an addition step of sexing to remove females before releasing.
Fu et al., (2010. PNAS, Vol. 107, No. 10, 4550-4554) discloses a method in which a fusion between RIDL system and a female sex-specific regulation based on an endogenous Actin-4 promoter that derived from Aedes aegypti females. The effector gene is specifically activated only in the direct-flight muscle of female mosquitoes and this expression makes females to be flightless. These females after eclosion would be stuck on the water surface and to be dead eventually. By this method, only 50% of offspring becomes males which can continuously pass the transformant genetic systems into next generation. However, this method in practice has a shortcoming. This happens when plenty of the flightless females staying on the water surface, their bodies and leg movements can prevent other eclosion males to come up with the water which may eventually drown males. The higher rearing density is the higher “collateral damage” for males, but in industrial insectary, rearing at high density is the only option.
Transformer-2 gene has been seen as a key factor in combination with Tra for sex determination in different eukaryotes although it may involve differently in different taxa depending on evolutionary divergence. Fortier and Belote (2000. Genesis 26(4): 240-244), Salvemini et al (2009. Int. J. Dev. Biol. 53: 109-120) and Sarno et al (2010. http://www.biomedcentral.com/1471-2148/10/140) used the RNAi method to knock down the Tra-2 genes in Drosophila, Ceratitis Capitata and Anastrepha, respectively. The knockdown effect can convert females of these species into pseudo males carrying XX chromosomes. In their studies, the RNA interference method is performed by injection of Tra-2 double stranded RNA (dsRNA) into embryos after an invitro synthesized step.
No tra-2 orthologue has been identified in Anopheles and the Tra-2 orthologue in Aedes aegypti mosquitoes seems to involve in a different genetic mode. A full length mRNA transcription of Tra-2 gene in Aedes aegypti is not necessarily required for its downstream gene cascade, doublesex (dsx) to be spliced. One female specific Dsx can be default spliced to be females (Salvemini et al., 2011. BMC Evolutionary Biology 2011, 11:41 http://www.biomedcentral.com/1471-2148/11/41). It, therefore, suggests that the wish to create all maleness offspring, including 50% pseudo [XX (mm)] males by a conversion from females is impossible if targeting Tra-2 in Culicinae. In fact, in our experiments, Tra-2 dsRNA injection into Culicinae mosquito eggs hasn't caused a significant bias in sex ratio.
The present invention sets out to overcome all the shortcoming of the previous methods by using the common principles of the RIDL method (Alphey, 1999. PN: WO 01/39599 A2; Alphey, 2007. Area-Wide Control of Insect Pests: From Research to Field Implementation, Springer, Dordrecht, The Netherlands) in combination with a discovery of X (m) bearing sperm killing effect due to Tra-2 RNAi genetic system. These transgenic Culicinae mosquitoes are therefore to produce more than 90% genetic maleness offspring.